Mitogen-activated Protein Kinase P38Modulates Pacemaker Ion Channels Differentiation In P19-derived Pluripotent Cells

Objective:In mammalian embryonal differentiation, morphogenesis of the cardiovascular system is initiated in20gestational days in humans and7.5gestational days in mice, followed by the formation of a simple tubular heart and generation of spontaneous beating. The generation of specific contractile proteins and ion channel proteins in earlier cardiac differentiation is triggered by cardiac-specific gene expression. It has recently been shown that the expression of cardiac genes is regulated by several specific transcription factors during embryonal heart differentiation. Csx/Nkx2.5, GATA4, and MEF2C are considered to be cardiac-specific transcription factors that play the critical roles in the early development of the heart, and serve as useful molecular markers to identify cardiac inductive signals from other tissues or germ layers.The homeobox gene Csx/Nkx2.5, a vertebrate homologue of Drosophila tinman, is one of the earliest known markers of mesoderm that give rise to myocardium, and its expression persists throughout the myocardium in the fully formed heart. GATA4serves as a cardiac-specific member of the GATA family of zinc finger transcription factors which is detected very early in the cardiogenetic area and persists later in the developing heart. MEF2C belongs to the MEF2family of MADS-box, and is expressed throughout the heart during mouse embryogenesis which controls cardiac morphogenesis and myogenesis. In the stimulation of cellular proliferation factors, including BMP, ET-1, and CT-1, the differentiation of cardiomyocytes is generated via the activation of intranuclear transcription factors such as Csx/Nkx2.5, GATA4, and MEF2C. However, in the early stage of embryonal development, signal transduction systems from the cellular membrane receptor signals into intranuclear transcription factors, subsequently activate the cardiac specific proteins including ion channel expression, are poorly understood. On the other hand, the MAP kinase pathways, ERK1/2, ERK5, JNK, and p38superfamilies, are major signalling systems by which the cells transfer extracellular signals into intracellular responses. It is known that MAP kinase performs the key roles in the differentiation of cardiomyocytes and in morphogenesis.Although several transcription factors are known to initial early cardiogenetic differentiation, their upstream signal regulators in responsible for the cellular automaticity are largely unknown. Our investigations were designed to clarify the role of transcription factors in P19-derived cardiomyocytes to the expression of cardiac pacemaker ion channels during the early phase of cardiogenetic differentiation, and their modulation by mitogen-activated protein (MAP) kinase cascade.Methods:P19cells were cardiogenetically differentiated by dimethyl sulfoxide into cardiomyocytes and three membrane ion channel currents responsible for pacemaker potentials, L-type Ca2+channel current (ICa.L), T-type Ca2+current (ICa.T), and hyperpolarization-activated inward current (If), were assessed by RT-PCR, Western blot, and patch clamp methods. Three MAP kinase superfamilies, p38, JNK and ERK1/2,5, were examined for their transcriptional modulation of the ion channels by use of distinct inhibitors.Results:During the early phase of cardiogenetic differentiation in P19-derived pluripotent cells, three transcription factors, Csx/Nkx2.5GATA4and MEF2C, known as initial markers for cardiac development, were all highly expressed as early as by day7prior to the spontaneous beating at day10. In the presence of a p38MAP kinase inhibitor SB203580, the spontaneous beating and the expression of three types of pacemaker ion channels were disrupted in P19-derived cardiomyocytes. Transcription factors Csx/Nkx2.5and GATA4but not MEF2C were markedly inhibited by p38MAP kinase inhibition in a distinct manner; expression but not phosphorylation of GATA4was reduced by inhibition of p38MAP kinase actions. On the other hand, in the presence of an ERK1/2,5inhibitor PD98059or a JNK MAP kinase inhibitor SP600125, P19cells successfully differentiated into cardiomyocytes displaying spontaneous beatings with expression of three types of pacemaker ion channels.Conclusions:We demonstrate that acquisition of cellular automaticity and the expression of pacemaker ion channels are regulated by the transcription factors, Csx/Nkx2.5and GATA4, through intracellular signals including p38MAP kinase in the process of P19-derived pluripotent cells differentiation into cardiomyocytes.